The long-range objective of this proposal is to study the regulation of glucose production (glycogenolysis and gluconeogenesis) both in vivo, using the conscious dog and in vitro using isolated hepatocytes. Gluconeogenesis will be studied by determining the hepatic uptake of alanine and lactate and by quantitating the conversion of 14C-alanine into 14C-glucose. Net hepatic glucose balance will be measured using arterio-venous (A-V) difference techniques while total glucose production will be measured using a tracer (3H-3-glucose) technique. These measurements will be made possible by surgical implantation of catheters for blood sampling in the portal and hepatic veins and femoral artery two weeks prior to study. Clear interpretation of data will be possible because both the pancreatic hormone levels and the plasma glucose concentrations will be controlled and will be precisely adjusted in accordance with the need of an experiment. Regulation of insulin and glucagon secretion will be achieved surgically or pharmacologically by inhibiting the animals' pancreatic hormone secretion and quantitatively replacing both insulin and glucagon intraportally. The surgical approach involves a "pancreatic autograft" technique and the pharmacological approach involves infusion of somatostatin (a potent inhibitor of insulin and glucagon secretion) coincident with pancreatic hormone replacement. Glucose will be regulated using a glucose-clamp technique. Utilizing the above methods our main objectives will be: 1) to examine the interaction of insulin and glucagon in regulating glucose production; 2) to examine the interaction between the plasma glucose level per se and insulin and glucagon in regulating glucose production; and 3) to more clearly define the time dependence of the action of glucagon and the role of "big" glucagon(s) on hepatic function. The last objective will also be examined using isolated hepatocytes where the mechanisms of the downregulation to glucagon and the action(s) of "big" glucagon(s) on enzymatic steps in the glycogenolytic cascade will be examined in detail.